Mutations in the macrophage scavenger receptor 1 gene alter risk of prostate cancer, asthma, and cardiovascular disease

ABSTRACT

The present invention discloses methods of screening a subject for increased likelihood or risk of certain diseases or disorders. This method comprises detecting the presence or absence of at least one mutation in the MSR1 gene wherein the presence or absence of such mutation indicates an increased risk for certain diseases, such as cancer asthma and/or cardiovascular diseases.

CROSS REFERENCE TO RELATED APPLICATION

The present application claims priority to and is a continuation of U.S.patent application Ser. No. 10/426,262, filed Apr. 30, 2003, now U.S.Pat. No. 7,579,147, which claims the benefit of U.S. ProvisionalApplication No. 60/378,377, filed May 7, 2002, the disclosure of each ofwhich is incorporated herein by reference in its entirety.

STATEMENT OF FEDERAL SUPPORT

This invention was made with government support under PHS SPORE CA58236and grants from the Department of Defense. The United States governmenthas certain rights to this invention.

FIELD OF THE INVENTION

This invention concerns gene mutations that alter the risk orsusceptibility to certain diseases, along with methods of use thereofand materials that may be used in such methods.

BACKGROUND OF THE INVENTION

Intense genetic study of familial prostate cancer has resulted in theidentification of numerous putative prostate cancer susceptibility lociand several candidate genes, along with a realization of the extensivegenetic and etiologic heterogeneity that characterizes this disease(Ostrander et al. (2000) Am. J. Hum. Genet. 67, 1367-1375). A gene orgenes on 8p22-23 have been implicated in prostate carcinogenesis by theobservation of frequent deletions of this region in prostate cancercells (Latil & Lidereau. (1998) Virchows Arch. 432, 389-406), and bythree recent linkage studies in hereditary prostate cancer (HPC)families (Xu et al. (2001) Am. J. Hum. Genet. 69, 341-350, Gibbs et al.(2000) Am. J. Hum. Genet. 67, 100-109, Goddard et al. (2001) Am. J. Hum.Genet 68, 1197-1206). Because it functions in multiple processesproposed to be relevant to prostate carcinogenesis (e.g. inflammation,innate and adaptive immunity, oxidative stress, and apoptosis) (De Marzoet al. (1999) Am. J. Pathol. 155, 1985-1992, Nelson et al. (2001)Urology 57, 39-45), the MSR1 (macrophage scavenger receptor) gene at8p22 is a promising candidate gene in this region (Platt & Gordon.(2001) J. Clin. Invest 108, 649-654).

SUMMARY OF THE INVENTION

Among other things, here we report the results of multiple geneticanalyses that indicate germline variants of MSR1 are associated withprostate cancer risk. In a mutation screen of MSR1 in germline DNAsamples from 159 HPC probands, one nonsense (R293X) and seven missensemutations (P36A, S41Y, V113A, D174Y, G369S, H441R and P275A) wereidentified in 11 families, including four of 14 African Americanfamilies studied. Additionally, we found a novel missense mutation,154V, in sporadic cases. A family-based linkage test providedstatistical evidence that these mutations co-segregate with prostatecancer (P=0.001); importantly, they were either not observed or observedless frequently in 274 men without prostate cancer and in 518 men whowere originally ascertained for a non-prostate related study. Sequenceanalyses predict that the nonsense change and a number of the missensechanges observed may significantly impact the function of the protein,and in some cases may impart dominant negative properties. Additionally,the allele frequencies of five other sequence variants in the coding,promoter, and intronic region of the gene are significantly differentbetween prostate cancer cases and controls (P=0.004). These resultsprovide genetic evidence that MSR1 plays an important role in prostatecancer susceptibility in Caucasians and African Americans, through bothrare mutations and more common sequence variants.

Accordingly, a first aspect of the present invention is a method ofscreening a subject for increased likelihood or risk of certain diseasesor disorders. The method comprises detecting the presence or absence ofat least one mutation in the MSR1 gene, the presence or absence of suchmutation indicating an increased risk of certain diseases.

MSR1 mutations of interest herein include, for example, the H441Rmutation, the G369S mutation, the R293X mutation, the P275A mutation,the DF174Y mutation, the V113A mutation, the S41Y mutation, the P36Amutation the 154V mutation, and other mutations which can be determinedby skilled persons in light of the information presented herein.

In one embodiment, the presence of an MSR1 mutation as described aboveindicates an increased risk of asthma in the subject, as compared tosubjects without the aforesaid mutation.

In another embodiment, the presence of an MSR1 mutation as describedabove indicates an increased risk of prostate cancer in said subject, ascompared to subjects without the said mutation.

In yet another embodiment, the absence of an MSR1 mutation as describedabove indicates an increased risk of cardiovascular disease in saidsubject, as compared to subjects with the said mutation (i.e., thepresence of an MSR 1 mutation as described above indicates a decreasedrisk of cardiovascular disease in said subjects, as compared to subjectswithout the said mutation).

A further aspect of the present invention is the use of a means ofdetecting an MSR1 mutation as described herein in determining if asubject is at increased or decreased risk for a disease as describedabove.

Still further aspects of the present invention include kits, reagentssuch as oligonucleotide probes, restriction enzymes and other such meansfor carrying out methods as described above, the use of such reagentsfor the preparation of kits or diagnostic reagents for carrying out themethods described above, the use of the MSR1 mutations described abovein structuring clinical trials of active agents for treating prostatecancer, asthma and/or cardiovascular disease as discussed above, and theuse of the MSR1 mutations described herein as targets for rational drugdesign.

The foregoing and other objects and aspects of the present invention areexplained in greater detail in the drawings herein and the specificationset forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a schematic of scavenger A receptor, with three isoforms.The predictive functional domains and corresponding position of aminoacid, as well as the results from protein alignment of multiple genomesis shown on the right side. The location of the rare mutationsidentified in the current study on the left side.

FIG. 2 shows the results of multipoint parametric linkage analyses ofprostate cancer susceptibility locus, using 24 markers on chromosome8p22-23 in the total 159 HPC families, as well as in the subset of 11families with the rare MSR1 mutations and 122 families without any ofthe rare mutations nor the frequent sequence variant (P275A).

FIG. 3 is a bar graph depicting total serum IgE in individualshomozygous for the wild-type MSR1, or heterzygous for the R293Xmutation.

FIG. 4 is a bar graph showing the frequency of the MSR1 nonsensemutation R293X in asthma patients as compared to controls.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is explained in greater detail below. Thisdescription is not intended to be a detailed catalog of all thedifferent ways in which the invention may be implemented, or all thefeatures that may be added to the instant invention. For example,features illustrated with respect to one embodiment may be incorporatedinto other embodiments, and features illustrated with respect to aparticular embodiment may be deleted from that embodiment. In addition,numerous variations and additions to the various embodiments suggestedherein will be apparent to those skilled in the art in light of theinstant disclosure which do not depart from the instant invention.Hence, the following specification is intended to illustrate someparticular embodiments of the invention, and not to exhaustively specifyall permutations, combinations and variations thereof.

Subjects for screening and/or treatment with the present invention are,in general, human subjects, including male and female subjects, withmale subjects preferred for the purpose of screening for prostate cancerrisk. The subject may be of any race and any age, including juvenile,adolescent, and adult, with adult subjects currently preferred. It willbe appreciated by those skilled in the art that, while the presentmethods are useful for screening subjects to provide an initialindication of the suitability of a patient for a particular treatment,this information will typically be considered by a clinician or medicalpractitioner in light of other factors and experience in reaching afinal judgment as to the treatment which any given subject shouldreceive.

Polymorphism detection. In general, the step of detecting thepolymorphism of interest may be carried out by collecting a biologicalsample containing DNA from the subject, and then determining thepresence or absence of DNA containing the polymorphism of interest inthe biological sample. Any biological sample which contains the DNA ofthat subject may be employed, including tissue samples and bloodsamples, with blood cells being a particularly convenient source. Thenucleotide sequence of the human MSR1 gene is known and suitable probes,restriction enzyme digestion techniques, or other means of detecting thepolymorphism may be implemented based on this known sequence inaccordance with standard techniques. See, e.g., U.S. Pat. Nos. 6,027,896and 5,767,248 to A. Roses et al. (Applicants specifically intend thatthe disclosures of all United States patent references cited herein beincorporated by reference herein in their entirety).

In describing peptides of this invention, the conventional andnon-conventional abbreviations for the various amino acids may be used.They are: Ala=A=Alanine; Val=V=Valine; Leu=L=Leucine; Ile=I=Isoleucine;Pro=P=Proline; Phe=F=Phenylalanine; Trp=W=Tryptophan; Met=M=Methionine;Gly=G=Glycine; Ser=S=Serine; Thr=T=Threonine; Cys=C=Cysteine; Tyr=Y=Tyrosine; Asn=N=Asparagine; Gln=Q=Glutamine; Asp=D=Aspartic Acid;Glu=E=Glutamic Acid; Lys=K=Lysine; Arg=R=Arginine; and His=H=Histidine.

The human macrophage scavenger receptor protein sequence andcorresponding nucleotide sequences are known. See, e.g., M. Emi et al.,J. Biol. Chem. 268 (3), 2120-2125 (1993); A. Matsumoto et al., Proc.Natl. Acad. Sci. USA 87(23), 9133-9137 (1990); NCBI Accession NumberP21757 (protein) and GenBank Accession Number D90187 (mRNA) (thedisclosures of which are to be incorporated herein by reference).

Thus, in one embodiment the amino acid sequence (SEQ ID NO: 2) of ahuman macrophage scavenger receptor protein I is:

  1 meqwdhfhnq qedtdscses vkfdarsmta llppnpknsp slqeklksfk aalialyllv 61 favlipligi vaaqllkwet kncsvsstna nditqsltgk gndseeemrf qevfmehmsn121 mekriqhild meanlmdteh fqnfsmttdq rfndillqls tlfssvqghg naideisksl181 islnttlldl qlnienlngk igentfkqqe eiskleervy nvsaeimamk eeqvhleqei241 kgevkvinni tndlrlkdwe hsqtlrnitl iqgppgppge kgdrgptges gprgfpgpig301 ppglkgdrga igfpgsrglp gyagrpgnsg pkgqkgekgs gntltpftkv rlvggsgphe361 grveilhsgq wgticddrwe vrvgqvvcrs lgypgvqavh kaahfgqgtg piwlnevfcf421 gressieeck irqwgtracs hsedagvtct l

In addition, in one embodiment the cDNA sequence (SEQ ID NO: 1) encodinghuman macrophage scavenger receptor protein I is as set forth below,with the translated sequence beginning at nucleotide 47:

   1 agagaagtgg ataaatcagt gctgctttct ttaggacgaa agaagtatgg agcagtggga  61 tcactttcac aatcaacagg aggacactga tagctgctcc gaatctgtga aatttgatgc 121 tcgctcaatg acagctttgc ttcctccgaa tcctaaaaac agcccttccc ttcaagagaa 181 actgaagtcc ttcaaagctg cactgattgc cctttacctc ctcgtgtttg cagttctcat 241 ccctctcatt ggaatagtgg cagctcaact cctgaagtgg gaaacgaaga attgctcagt 301 tagttcaact aatgcaaatg atataactca aagtctcacg ggaaaaggaa atgacagcga 361 agaggaaatg agatttcaag aagtctttat ggaacacatg agcaacatgg agaagagaat 421 ccagcatatt ttagacatgg aagccaacct catggacaca gagcatttcc aaaatttcag 481 catgacaact gatcaaagat ttaatgacat tcttctgcag ctaagtacct tgttttcctc 541 agtccaggga catgggaatg caatagatga aatctccaag tccttaataa gtttgaatac 601 cacattgctt gatttgcagc tcaacataga aaatctgaat ggcaaaatcc aagagaatac 661 cttcaaacaa caagaggaaa tcagtaaatt agaggagcgt gtttacaatg tatcagcaga 721 aattatggct atgaaagaag aacaagtgca tttggaacag gaaataaaag gagaagtgaa 781 agtactgaat aacatcacta atgatctcag actgaaagat tgggaacatt ctcagacctt 841 gagaaatatc actttaattc aaggtcctcc tggacccccg ggtgaaaaag gagatcgagg 901 tcccactgga gaaagtggtc cacgaggatt tccaggtcca ataggtcctc cgggtcttaa 961 aggtgatcgg ggagcaattg gctttcctgg aagtcgagga ctcccaggat atgccggaag1021 gccaggaaat tctggaccaa aaggccagaa aggggaaaag gggagtggaa acacattaac1081 tccatttacg aaagttcgac tggtcggtgg gagcggccct cacgagggga gagtggagat1141 actccacagc ggccagtggg gtacaatttg tgacgatcgc tgggaagtgc gcgttggaca1201 ggtcgtctgt aggagcttgg gatacccagg tgttcaagcc gtgcacaagg cagctcactt1261 tggacaaggt actggtccaa tatggctgaa tgaagtgttt tgttttggga gagaatcatc1321 tattgaagaa tgtaaaattc ggcaatgggg gacaagagcc tgttcacatt ctgaagatgc1381 tggagtcact tgcactttat aatgcatcat attttcattc acaactatga aatcgctgct1441 caaaaatgat tttattacct tgttcctgta aaatccattt aatcaatatt taagagatta1501 agaatattgc ccaaataata ttttagatta caggattaat atattgaaca ccttcatgct1561 tactatttta tgtctatatt taaatcattt taacttctat aggtttttaa atggaatttt1621 ctaatataat gacttatatg ctgaattgaa cattttgaag tttatagctt ccagattaca1681 aaggccaagg gtaatagaaa tgcataccag taattggctc caattcataa tatgttcacc1741 aggagattac aattttttgc tcttcttgtc tttgtaatct atttagttga ttttaattac1801 tttctgaata acggaaggga tcagaagata tcttttgtgc ctagattgca aaatctccaa1861 tccacacata ttgttttaaa ataagaatgt tatccaacta ttaagatatc tcaatgtgca1921 ataacttgtg tattagatat caatgttaat gatatgtctt ggccactatg gaccagggag1981 cttatttttc ttgtcatgta ctgacaactg tttaattgaa tcatgaag

In describing the mutations disclosed herein in the novel proteinsdescribed herein, and the nucleotides encoding the same, the namingmethod is as follows: [amino acid replaced] [amino acid number insequence of known protein][alternate amino acid]. For example, for theH441R variant disclosed herein, histidine at the 441^(st) amino acid inthe protein is replaced with arginine.

The polymorphisms described herein can be detected in accordance withknown techniques based upon the known sequence information of the humanMSR1 gene and the information provided herein. Novel nucleic acidsequences and proteins described herein can be isolated from humansources based upon the information provided herein or produced by othermeans such as site-directed mutagenesis of known or available nucleicacids, coupled as necessary with techniques for the production ofrecombinant proteins known in the art.

Determining the presence or absence of DNA containing a polymorphism ormutation of interest may be carried out with an oligonucleotide probelabeled with a suitable detectable group, or by means of anamplification reaction such as a polymerase chain reaction or ligasechain reaction (the product of which amplification reaction may then bedetected with a labeled oligonucleotide probe or a number of othertechniques). Further, the detecting step may include the step ofdetecting whether the subject is heterozygous or homozygous for thepolymorphism of interest. Numerous different oligonucleotide probe assayformats are known which may be employed to carry out the presentinvention. See, e.g., U.S. Pat. No. 4,302,204 to Wahl et al.; U.S. Pat.No. 4,358,535 to Falkow et al.; U.S. Pat. No. 4,563,419 to Ranki et al.;and U.S. Pat. No. 4,994,373 to Stavrianopoulos et al. (applicantsspecifically intend that the disclosures of all U.S. Patent referencescited herein be incorporated herein by reference).

Amplification of a selected, or target, nucleic acid sequence may becarried out by any suitable means. See generally D. Kwoh and T. Kwoh,Am. Biotechnol. Lab. 8, 14-25 (1990). Examples of suitable amplificationtechniques include, but are not limited to, polymerase chain reaction,ligase chain reaction, strand displacement amplification (see generallyG. Walker et al., Proc. Natl. Acad. Sci. USA 89, 392-396 (1992); G.Walker et al., Nucleic Acids Res. 20, 1691-1696 (1992)),transcription-based amplification (see D. Kwoh et al., Proc. Natl. AcadSci. USA 86, 1173-1177 (1989)), self-sustained sequence replication (or“3SR”) (see J. Guatelli et al., Proc. Natl. Acad. Sci. USA 87, 1874-1878(1990)), the QB replicase system (see P. Lizardi et al., BioTechnology6, 1197-1202 (1988)), nucleic acid sequence-based amplification (or“NASBA”) (see R. Lewis, Genetic Engineering News 12 (9), 1 (1992)), therepair chain reaction (or “RCR”) (see R. Lewis, supra), and boomerangDNA amplification (or “BDA”) (see R. Lewis, supra).

DNA amplification techniques such as the foregoing can involve the useof a probe, a pair of probes, or two pairs of probes which specificallybind to DNA containing the polymorphism of interest, but do not bind toDNA that does not contain the polymorphism of interest under the samehybridization conditions, and which serve as the primer or primers forthe amplification of the DNA or a portion thereof in the amplificationreaction. Such probes are sometimes referred to as amplification probesor primers herein.

In general, an oligonucleotide probe which is used to detect DNAcontaining a polymorphism or mutation of interest is an oligonucleotideprobe which binds to DNA encoding that mutation or polymorphism, butdoes not bind to DNA that does not contain the mutation or polymorphismunder the same hybridization conditions. The oligonucleotide probe islabeled with a suitable detectable group, such as those set forth belowin connection with antibodies. Such probes are sometimes referred to asdetection probes or primers herein.

Probes and primers, including those for either amplification and/orprotection, are nucleotides (including naturally occurring nucleotidessuch as DNA and synthetic and/or modified nucleotides) are any suitablelength, but are typically from 5, 6, or 8 nucleotides in length up to40, 50 or 60 nucleotides in length, or more. Such probes and or primersmay be immobilized on or coupled to a solid support such as a bead,chip, pin, or microtiter plate well in accordance with known techniques,and/or coupled to or labeled with a detectable group such as afluorescent compound, a chemiluminescent compound, a radioactiveelement, or an enzyme in accordance with known techniques.

Polymerase chain reaction (PCR) may be carried out in accordance withknown techniques. See, e.g., U.S. Pat. Nos. 4,683,195; 4,683,202;4,800,159; and 4,965,188. In general, PCR involves, first, treating anucleic acid sample (e.g., in the presence of a heat stable DNApolymerase) with one oligonucleotide primer for each strand of thespecific sequence to be detected under hybridizing conditions so that anextension product of each primer is synthesized which is complementaryto each nucleic acid strand, with the primers sufficiently complementaryto each strand of the specific sequence to hybridize therewith so thatthe extension product synthesized from each primer, when it is separatedfrom its complement, can serve as a template for synthesis of theextension product of the other primer, and then treating the sampleunder denaturing conditions to separate the primer extension productsfrom their templates if the sequence or sequences to be detected arepresent. These steps are cyclically repeated until the desired degree ofamplification is obtained. Detection of the amplified sequence may becarried out by adding to the reaction product an oligonucleotide probecapable of hybridizing to the reaction product (e.g., an oligonucleotideprobe of the present invention), the probe carrying a detectable label,and then detecting the label in accordance with known techniques, or bydirect visualization on a gel. When PCR conditions allow foramplification of all allelic types, the types can be distinguished byhybridization with allelic specific probe, by restriction endonucleasedigestion, by electrophoresis on denaturing gradient gels, or othertechniques.

Ligase chain reaction (LCR) is also carried out in accordance with knowntechniques. See, e.g., R. Weiss, Science 254, 1292 (1991). In general,the reaction is carried out with two pairs of oligonucleotide probes:one pair binds to one strand of the sequence to be detected; the otherpair binds to the other strand of the sequence to be detected. Each pairtogether completely overlaps the strand to which it corresponds. Thereaction is carried out by, first, denaturing (e.g., separating) thestrands of the sequence to be detected, then reacting the strands withthe two pairs of oligonucleotide probes in the presence of a heat stableligase so that each pair of oligonucleotide probes is ligated together,then separating the reaction product, and then cyclically repeating theprocess until the sequence has been amplified to the desired degree.Detection may then be carried out in like manner as described above withrespect to PCR.

It will be readily appreciated that the detecting steps described hereinmay be carried out directly or indirectly. For example, a polymorphismor mutation could be detected by measuring by digestion with restrictionenzymes, detection of markers that are linked to the mutation orpolymorphism, etc.

Kits useful for carrying out the methods of the present invention will,in general, comprise one or more oligonucleotide probes and otherreagents for carrying out the methods as described above, such asrestriction enzymes, optionally packaged with suitable instructions forcarrying out the methods.

The new polymorphisms described herein provide novel nucleic acidsencoding the human MSR1, along with probes such as described above thatbind selectively thereto. Such nucleic acids can be inserted intovectors such as plasmids, optionally associated with or placed under thecontrol of a promoter, and the nucleic acids may be inserted into hostcells and optionally expressed therein (when the promoter is operativein the host cell) to produce MSR1.

The present invention also provides a method of conducting a clinicaltrial on a plurality of human subjects or patients. Such methodsadvantageously permit the refinement of the patient population so thatadvantages of particular treatment regimens (typically administration ofpharmaceutically active organic compound active agents) can be moreaccurately detected, particularly with respect to particularsub-populations of patients. In general, such methods compriseadministering a test active agent or therapy to a plurality of subjects(a control or placebo therapy typically being administered to a separatebut similarly characterized plurality of subjects) and detecting thepresence or absence of at least one mutation or polymorphism asdescribed above in the plurality of subjects. The polymorphisms may bedetected before, after, or concurrently with the step of administeringthe test therapy. The influence of one or more detected polymorphisms orabsent polymorphisms on the test therapy can then be determined on anysuitable parameter or potential treatment outcome or consequence,including but not limited to: the efficacy of said therapy, lack of sideeffects of the therapy, etc.

The present invention is explained in greater detail in the followingnon-limiting examples.

Example 1 Methods

Subjects. Study subjects were from four different populations. The firstsubjects were 159 HPC families ascertained at the Brady UrologyInstitute at Johns Hopkins Hospital (Baltimore, Md.), through referrals,review of medical records for patients seen at Johns Hopkins Hospitalfor treatment of prostate cancer, and respondents to various laypublications describing our studies (Xu et al. (2001) Hum. Genet. 108,335-345). Each family had at least three first-degree relatives affectedwith prostate cancer. The diagnosis of prostate cancer was then verifiedby medical records. The mean age at prostate cancer diagnosis for theseprobands was 61 years; 133 (84%) were Caucasian, and 14 (8.8%) wereAfrican American. The second was a case-control population. Two hundredand forty-six sporadic cases were recruited from patients that underwenttreatment for prostate cancer at the Johns Hopkins Hospital and did nothave first-degree relatives affected with prostate cancer. Among them,233 are Caucasians and 13 are African Americans. For each subject, thediagnosis of prostate cancer was confirmed by pathology reports. Meanage at diagnosis for these cases was 58.6 years. Two hundred andthirty-nine non-prostate cancer controls were recruited among menparticipating in screening programs for prostate cancer who had normaldigital rectal examination (DRE) and normal PSA levels (i.e., <4 ng/ml),consisting of 164 Caucasians and 75 African Americans. The mean age atexamination was 57.5 years. The third group was a small African Americancase-control population from Wake Forest University School of Medicine.This population was added to this study to enlarge the sample size ofAfrican Americans. Among them, 31 were prostate cancer cases and 20 wereunaffected controls that participated in screening programs, who were atleast 50 years of age and had normal DRE and PSA levels. The last was asubset (n=518) of a large population study of asbestos-exposed workerswho were recruited to study the impact of genetic and environmentalfactors on the development of asbestos-induced lung diseases. Serum PSAlevels and prostate cancer diagnoses were later obtained. Participantsworked as painters, pipefitters, plumbers, operators, and electricians.A physical examination was performed on all participants. TheInstitutional Review Boards of Johns Hopkins University, St. LouisUniversity, and Wake Forest University approved each of the studyprotocols.

Sequencing methods and SNP genotyping. The primers for PCR are availableupon request. All PCR reactions were performed in a 10 μl volumeconsisting of 30 ng genomic DNA, 0.2 μM of each primer, 0.2 mM of eachdNTP, 1.5 mM MgCl₂, 20 mM Tris-HCl, 50 mM KCl, and 0.5 units Taqpolymerase (Life Technologies, Inc.). PCR cycling conditions were asfollows: 94° C. for 4 minutes; followed by 30 cycles of 94° C. for 30seconds, specified annealing temperature for 30 seconds, and 72° C. for30 seconds; with a final extension of 72° C. for 6 minutes. All PCRproducts were purified using the QuickStep™ PCR purification Kit (EdgeBioSystems, Gaithersburg, Md.) to remove dNTPs and excess primers. Allsequencing reactions were performed using dye-terminator chemistry(BigDye, ABI, Foster City, Calif.) and then precipitated using 63+/−5%ethanol. Samples were loaded onto an ABI 3700 DNA Analyzer after adding8 μl of formamide. SNPs were identified using Sequencher™ softwareversion 4.0.5 (Gene Codes Corporation).

Computational Analysis. Complete human mRNA sequence corresponding toMSR1 Type I and II isoforms was assembled by optimal pairwise alignmentof mRNA sub-sequences using the GCG Bestfit program (Accelrys). Only thecoding sequence of Type III was available in GenBank. Exon-intronboundaries in NCBI human genome chromosome 8 sequence were delineated bySmith-Waterman alignment of assembled Type I, II, III mRNA sequences tothe human genome sequence using the Swat program (P. Green,unpublished). Secondary structure protein analysis was performed usingGCG programs. Transmembrane domain prediction was performed using HMMTOP2.0 (Tusnady & Simon. (2001) Bioinformatics. 17, 849-850) and TMHMM 2.0(Krogh et al. (2001) J. Mol. Biol. 305, 567-580).

Accession Numbers. Nucleotide: D13263 Human MSR1 promoter and exon 1;D90187 Type I mRNA coding sequence; D13264 Type 13′ UTR sequence; D90188Type II mRNA coding sequence; D13265 Type II 3′ UTR sequence; AF037351Type III coding sequence. Peptide: BAA14298 MSR1 Type I proteinsequence; BAA14299 MSR1 Type II protein sequence; AAC09251 Type IIIprotein sequence. Genomic: NT_(—)015280.5 human genome chromosome 8sequence contig.

Statistical analysis. Hardy-Weinberg Equilibrium (HWE) tests for allSNPs, and linkage disequilibrium (LD) tests for all pairs of SNPs, wereperformed using the GDA computer program (Weir. (1996) Genetic DataAnalysis II: Methods for Discrete Population Genetic Data. Sinauer,Sunderland, Mass.). Linkage analyses were performed using bothparametric and non-mode-of-inheritance methods, implemented by thecomputer program GENEHUNTER (Kruglyak et al. (1996) Am. J. Hum. Genet.58, 1347-1363). For the parametric analysis, the same autosomal dominantmodel that was used by Smith et al. (1996) Science 274, 1371-1374 wasassumed. Linkage in the presence of heterogeneity was assessed by use ofSmith's admixture test for heterogeneity (Ott. (1998) Analysis of humangenetic linkage (3rd ed). Johns Hopkins Press, Baltimore, Md.). Amaximum likelihood approach was used to estimate the proportion oflinked families (α), by maximizing the admixed LOD score (HLOD). Alikelihood ratio test was used to test for different proportion oflinked families (α's) between two groups of families, and aχ²=4.6×(HLOD+HLOD₂−HLOD_(total)) is calculated which has 1 degree offreedom, where HLOD₁, HLOD₂, and HLOD_(total) are the HLODs for the twosubsets of familie and the whole sample, respectively. For thenon-mode-of-inheritance analysis, a statistic ‘Z-all’ in the program wasused (Whittemore & Halpern. (1994) Biometrics 50, 109-117). To test forco-segregation between the rare mutations and prostate cancer, weconstructed a bi-allelic marker by coding all 7 different rare mutationsinto one mutation. Family-based linkage and association tests wereperformed using FBAT software (Lissbrant et al. (2000) Int. J. Oncol.17, 445-451). Utilizing data from nuclear families and sibships, FBATdetermines an S statistics from the data, which is the linearcombination of offspring genotypes and phenotypes. The distribution ofthe S statistics is generated by treating the offspring genotype data asrandom, and conditioning on the phenotypes and parental genotypes. A Zstatistics and its corresponding p-value are calculated. The hypothesesof differences in allele frequencies between cases and controls weretested based on the χ² of Amitage trend tests (Sasieni. (1997)Biometrics 53, 1253-1261, Slager & Schaid. (2001) Hum. Hered. 52,149-153), adjusting for age.

Example 2 Results

The present invention also illustrates evidence for a prostate cancerlinkage at 8p22-23 in a study of 159 HPC families, each with at leastthree affected first-degree relatives (Xu et al. (2001) Am. J. Hum.Genet. 69, 341-350). Evidence for linkage at this region was alsoobserved in two other HPC family studies (Gibbs, M. et al. (2000) Am. J.Hum. Genet. 67, 100-109, Goddard et al. (2001) Am. J. Hum. Genet. 68,1197-1206). Together with the well-known observations that 8p is themost frequent site of loss of heterozygosity (LOH) in prostate cancercells (Latil & Lidereau. (1998) Virchows Arch. 432, 389-406), theseresults implicate a prostate cancer susceptibility locus at 8p22-23. Toidentify prostate cancer susceptibility gene(s), known genes andtranscripts in this region are being systemically screened andprioritized by biological relevance.

MSR1 is one of two known genes located in a previously characterizedhomozygous deletion in a clinical prostate cancer sample (Bova et al.(1993) Cancer Res. 53, 3869-3873). The MSR1 protein, a Class A scavengerreceptor (SR-A), is a multi-domain trimeric molecule composed ofidentical protein chains. It has two functional isoforms (Type I and II)and one nonfunctional isoform (III), generated by alternative splicingof a single 11-exon mRNA (Matsumoto et al. (1990) Proc. Natl. Acad. Sci.U.S.A 87, 9133-9137, Emi et al. (1993) J. Biol. Chem. 268, 2120-2125).This macrophage-specific receptor is capable of binding a highly diversearray of polyanionic ligands, ranging from gram negative and positivebacteria, oxidized LDL, to silica, and correspondingly has been linkedto a wide variety of normal and pathologic processes includinginflammation, innate and adaptive immunity, oxidative stress, andapoptosis (Platt & Gordon. (2001) J. Clin. Invest 108, 649-654). Thisgene presented an important candidate for analysis because of recenthypotheses concerning the mechanisms of prostate carcinogenesisimplicate some or all of these processes and the degree of macrophageinfiltration have been associated with prostate cancer prognosis inrecent studies (Shimura et al. (2000) Cancer Res. 60, 5857-5861,Lissbrant et al. (2000) Int. J. Oncol. 17, 445-451).

To evaluate the role of MSR1 in prostate cancer a comprehensive geneticstudy was performed in a large number of subjects from multiplepopulations. These analyses consisted of: 1) sequencing the entirecoding region of the gene in 159 HPC probands; this analysis resulted inthe identification of multiple rare mutations as well as common sequencevariants; 2) genotyping the 7 identified mutations in all family membersof the 159 HPC families to examine co-segregation between the mutationsand prostate cancer; 3) genotyping the 7 mutations in additional 276sporadic cases and 274 unaffected controls to estimate their frequenciesin non-hereditary prostate cancer cases and controls; 4) genotyping the7 mutations in 518 men who were not ascertained for their prostatecancer status to evaluate the mutation frequencies in the generalpopulation; and 5) genotyping the 6 identified, frequently observedsequence variants in the case-control population to test for associationwith prostate cancer.

Mutations and sequence variants of MSR1 were first screened in thegermline DNA samples of one affected individual (proband) from each ofthe 159 HPC families. The PCR products of all 11 exons, exon-intronjunctions, promoter region, 5′ and 3′ UTRs, were directly sequenced.Eight nonsynonymous changes were identified (Table 1 and FIG. 1),including one nonsense mutation at codon 293 (R293X) and seven missensemutations or sequence variants (P36A, S41Y, V113A, D174Y, P275A, G369S,H441R). Although over 130 and 100 SNPs are listed in NCBI's dbSNPdatabase and Celera's Human RefSNP database, respectively, for the MSR1gene, none of these reported sequence changes lie in the coding region.Since the sequence variants observed had not been previously reported, astudy was performed to investigate the co-segregation of these mutationswith prostate cancer. In doing so, all family members with available DNAsamples among the 159 HPC families (n=1477, including 653 affectedindividuals) were directly sequenced for these mutations.

Sequence variant R293X. The nonsense mutation in Exon 6 (R293X) wasobserved in four different families, all of which are Caucasian (Table1, for reasons of confidentiality, pedigrees were not drawn). Themutation segregates well, although not completely, with prostate cancerin these nuclear families. Eight of the 9 affected brothers in thesefamilies had the mutation. The only affected brother who did not havethe mutation (in family 30) was diagnosed with prostate cancer at age 78years, being screened as a result of his family history, and is stillalive, while two of his affected brothers died of prostate cancer. Ofthe two unaffected brothers from whom blood was available in thesefamilies, one carried the mutation; this individual was diagnosed withcolon cancer at age 70.

Sequence variant D174Y. The missense change (D174Y) in Exon 4 wasobserved in four families, all African American (there were 14 AfricanAmerican families in our study). Again, the mutation segregates well,but not completely, with prostate cancer in these families (Table 1).Seven of the 9 affected brothers in these families had the mutation,while one unaffected brother also had the mutation; however, thisunaffected brother is under 50 years of age. Family 150 providedparticularly strong evidence for co-segregation between the mutation andprostate cancer. All six affected men in the family had the mutation,including four brothers, a half paternal brother and his affected son.The half brother's unaffected son did not have the mutation.

TABLE 1 Description of the 11 families with mutations in MSR1 No. withmutation/total probable Brothers Brothers transmission Mutation FamilyRace with HPC w/o HPC of mutation** Additional information R293X 30Caucasian 2/3 1*/1  Unknown *The unaffected brother had colon cancer,three other siblings died of either prostate, lung, or breast cancer. 91Caucasian 1/1 0/0 Maternal Mother had breast cancer and father isunaffected. None of the extended paternal relatives had the mutation.133 Caucasian 2/2 0/0 Paternal Father had rectal cancer, none of theextended maternal relatives, including 4 unaffecteds had the mutation.223 Caucasian 3/3 0/1 Maternal None of the extended paternal relativehad the mutation. D174Y 48 African 1/2 0/0 Paternal Father is anobligate Am. carrier, 7 out of 10 paternal siblings had prostate, lung,ovarian, and colon cancers. 51 African 1/1 0/0 Maternal Mother is amutation Am. carrier. 150 African 4/4 0/0 Paternal Paternal half brotherand Am. his affected son had the mutation, the unaffected son did nothave the mutation. 165 African 1/2 1/1 Unknown A mutation carrier sisterAm died of breast cancer, the unaffected brother is under 50. P36A 118Caucasian 3/3 0/0 Unknown One of the affected brothers and anotherbrother had melanoma. V113A 118 Caucasian 3/3 0/0 Unknown All threemutation carriers had mutation P36A. S41Y 51 African 1/1 0/0 MaternalThe mutation carrier had Am the mutation D174Y. G369S 196 Caucasian 3/31/1 Unknown The unaffected brother is over 70 years old. 441R 65Caucasian 4/4 1/1 Maternal The unaffected brother is under 60 years old,mother had breast cancer, none of the extended paternal relatives hadthe mutation. Total 29/32 4/5 **Primarily based on the haplotype andmutation carrier status in the paternal or maternal relatives

Additional sequence variants. Five additional missense mutations wereobserved in four independent probands. All 11 affected brothers in thesefour families had the same mutation found in the corresponding proband,although two unaffected brothers also had mutations (Table 1). Family118 had two mutations (P36A and V113A); however, it is unclear whetherthese two mutations are on the same chromosome because all threeaffected brothers shared both parental chromosomes at this locus. Theaffected individual in Family 51 also had two mutations (S41Y and D174Y)inherited on the same maternal chromosome. Overall, the combinedanalyses for these seven mutations demonstrated that 29 out of 32affected sons in these nuclear families had the mutation present intheir family. This observation is of interest considering thecomplexities of the disease, including the significant degree of locusand allelic heterogeneity, phenocopies, and incomplete penetrance. Sevenout of these 11 (64%) families had other types of cancer in theirfirst-degree relatives; this is however not significantly higher thanthe rate in the remaining families (53%).

To formally test for co-segregation between these mutations and prostatecancer in the 159 HPC families a family-based linkage and associationanalysis was performed as implemented in FBAT computer program (Laird etal. (2000) Genet. Epidemiol. 19 Suppl 1, S36-S42), using the status ofmutations as a bi-allelic marker. A higher than expected S teststatistic was observed for the combined mutant allele (Z=3.17, P=0.001),providing significant evidence for linkage between these mutations andprostate cancer. This result is consistent with the findings frommultiple segregation analyses that a rare but highly penetrant autosomaldominant allele segregates in some prostate cancer families (Carter etal. (1992) Proc. Natl. Acad. Sci. U.S.A 89, 3367-3371, Gronberg et al.(1997) Am. J. Epidemiol. 146, 552-557, Schaid et al. (1998) Am. J. Hum.Genet. 62, 1425-1438, Cui et al. (2001) Am. J. Hum. Genet. 68,1207-1218.

To examine the relationship between MSR1 mutations and previouslyobserved evidence for linkage of prostate cancer to 8p22-23 (Xu et al.(2001) Am. J. Hum. Genet. 69, 341-350) was re-analyzed the linkage datawas re-analyzed using the same 24 markers, comparing two sets offamilies: the 11 families with any of the MSR1 mutations described aboveand the 122 families without any of these mutations nor the frequentmissense mutation P275A (see below). The group of 11 families withmutations provided notably higher LOD scores in this region than the 122families without mutations (FIG. 2), suggesting that the former familiescontribute disproportionately to the overall linkage at 8p22-23. Moreinterestingly, the maximum LOD score assuming heterogeneity (HLOD) forthese 11 families was 1.40 (P=0.01) at MSR1 region. The proportion offamilies (a) linked to the locus D8S1135, the closest microsatellitemarker to MSR1, was significantly higher in the 11 families (45%) thanin the 122 families (2%), with a χ₁ ²=4.28 (P=0.038). Six of the 11mutation carrying families had positive LOD scores at D8S1135, includingtwo families with LOD scores >0.6, and two with LOD >1.0. The remainingfive families had negative LOD scores at this locus, possibly due tolocus heterogeneity within extended families. For example, in Family133, while both affected brothers in one sib-ship carry the haplotypebearing the R293X mutation, none of their 11 genotyped maternalrelatives in five other branches (including four men affected withprostate cancer) carried this haplotype or the corresponding mutation.Consequently, parametric linkage analysis provided a LOD score of −1.8for this family. Further examination indicates that the MSR1 mutation inthis family was transmitted from the married-in father of the twomutation carriers. This individual did not have prostate cancer; he wasdiagnosed with rectal cancer before age 60.

To estimate the frequencies of these mutations in non-hereditaryprostate cancer patients and unaffected men, additional sporadicprostate cancer patients were screened (233 Caucasians and 43 AfricanAmericans) along with unaffected controls (164 Caucasians and 110African Americans). As shown in Table 2, the mutations P36A and G369Swere not found in any Caucasian or African American case-controlsubjects. The mutation H441R was only found one time, in a Caucasiansporadic prostate cancer cases (0.4%). The mutation S41Y was found onetime each in a Caucasian case (0.4%), an African American case (2.3%),and an African American unaffected control (0.9%). This unaffectedcontrol carrier has a positive family history of prostate cancer. Theother two mutations (R293X and D174Y) were found multiple times in thesesubjects. The nonsense mutation (R293X) was only found in Caucasiansubjects and was more often observed in cases (n=3, 1.3%) than incontrols (n=1, 0.6%). This control carrier is 65 years old and has aserum PSA level of 2.1 ng/ml. The missense mutation D174Y was primarilyobserved in African American subjects and was found more often in cases(n=3, 7.0%) than in unaffected controls (n=2, 1.8%). Both of theseunaffected control mutation carriers (ages 56 and 60) have a positivefamily history of prostate cancer, although their PSA values are normal.The only Caucasian carrier of the mutation D 174Y is a case.Interestingly, all four S41Y mutation carriers (three African Americanand one Caucasian) also have the mutation D174Y, suggesting a foundereffect. Overall, these results suggest that the identified mutations arelow frequency and potentially high penetrance. The observation ofmutations in some unaffected controls is not surprising consideringprostate cancer is a late age of onset disease.

TABLE 2 Summary of the mutations or sequence variants in the codingregion of MSR1 Number (frequency) of subjects with mutations or sequencevariants¹ P36A S41Y V113A D174Y P275A R293X G369S H441R Exon 3 3 4 4 6 610 11 Nucleotide C/G C/A T/C G/T C/G C/T G/A A/G change Caucasians HPCfamilies 1 (0.7%) 0 (0.0%) 1 (0.7%) 0 (0.0%) 28 (21.0%) 4 (3.0%) 1(0.7%) 1 (0.7%) (n = 133) HPC probands 1 (0.7%) 0 (0.0%) 1 (0.7%) 0(0.0%) 16 (12.2%) 2* (1.5%)  1 (0.7%) 1 (0.7%) (n = 133) Sporadic cases0 (0.0%) 1 (0.4%) 2 (0.8%) 1 (0.4%) 19 (8.1%)  3 (1.3%) 0 (0.0%) 1(0.4%) (n = 233) Unaffected 0 (0.0%) 0 (0.0%) 1 (0.6%) 0 (0.0%) 29(17.7%) 1 (0.6%) 0 (0.0%) 0 (0.0%) controls (n = 164) Asbestos 0 7**(1.5%)  exposed men (n = 469) African Americans HPC families 0 (0.0%) 1(7.1%) 0 (0.0%)  4 (28.6%)  2 (14.3%) 0 (0.0%) 0 (0.0%) 0 (0.0%) (n =14) HPC probands 0 (0.0%) 1 (7.1%) 0 (0.0%) 2* (14.3%) 1 (7.1%) 0 (0.0%)0 (0.0%) 0 (0.0%) (n = 14) Sporadic cases 0 (0.0%) 1 (2.3%) 0 (0.0%) 3(7.0%)  5 (11.6%) 0 (0.0%) 0 (0.0%) 0 (0.0%) (n = 43) Unaffected 0(0.0%) 1 (0.9%) 0 (0.0%) 2 (1.8%) 12 (10.9%) 0 (0.0%) 0 (0.0%) 0 (0.0%)controls (n = 110) Asbestos 2 (4.2%) 0 (0.0%) exposed men (n = 49)

To begin to understand the frequency and the impact of the mutationsR293X and D174Y in the general population 518 men were further screenedwho were originally ascertained for an asbestos study, regardless oftheir prostate cancer status. The diagnosis of prostate cancer and serumPSA levels was determined subsequently. The racial distribution of thesubjects was 91% Caucasians and 9% African Americans. The mean age ofthe study subjects at examination was 63.3 years. A diagnosis ofprostate cancer was reported by 5.8% (n=30) of men, and this rate wassimilar in both Caucasians (5.8%, n=27) and African Americans (6.1%,n=3). The nonsense mutation (R293X) was observed 7 times among 469Caucasians (1.5%) in this group (Table 2). One of these men was amongthe 27 men subsequently diagnosed with prostate cancer (3.7%). Anothernonsense mutation carrier had his prostate removed, and a correspondingundetectable serum PSA level, although his diagnosis of prostate cancerwas not confirmed. Two other men had elevated PSA levels (11.8 and 4.2ng/ml) although they were elderly (ages 72 and 76 years, respectively).No biopsy data were available for these two men. Three other men did notreport a diagnosis of prostate cancer and had normal PSA levels (1.5ng/mL at age 58, 0.8 ng/mL at age 62, and 1.8 ng/mL at age 74). Overall,this mutation was found among 4 of 72 men in this population (5.6%) whohave either a diagnosis of prostate cancer or PSA levels outside thenormal range (≧4 ng/mL or ≦0.2 ng/mL), as compared to 3 of the 397 men(0.7%) without a diagnosis of prostate cancer and normal PSA levels. Thedifference in mutation carrier rates between these two groups isstatistically significant (Fisher Exact Test, P=0.01), suggesting themutation carriers have an increased risk for prostate pathology(OR=7.72, 95% confidence interval (CI) 1.69-35.2). The missense mutation(D174Y) was observed two times in 49 unaffected African American men(4.1%). One has a PSA of 3.4 ng/mL at age 64 and the other is 43 yearsof age.

Besides these relatively rare mutations in the coding regions, six othersequence variants were frequently observed in the 159 HPC probands(Table 3), including SNPs in the coding region of Exon 6 (P275A),promoter region (PROd), Intron 5 (15b), and Intron 6 (16a), and aninsertion/deletion in Intron 1 (INDEL1), and Intron 7 (INDEL7). BecauseP275A is an exonic SNP, it was screened among all the family memberswith available DNA samples in the 159 HPC families. Thirty families hadat least one family member who had the variant allele ‘G’; of these, 28are Caucasian families and 2 are African American families. Tests forlinkage between this SNP and prostate cancer in these families provideda low LOD score (two-point maximum LOD was 0.27 at θ of 0.26 using adominant model) and a low NPL Z-score (0.55, P=0.28). A family-basedlinkage and association test provided no evidence for linkage, with alower than expected observed S statistic for the variant allele ‘G’(Z=0.12, P=0.90). This data provide scant evidence that this SNPsegregates with prostate cancer in these families.

TABLE 3 Sequence variants of MSR1 and their frequencies in cases andcontrols (Caucasians only) Genotype frequencies SNPs HPC SporadicControls P-values (vs. controls)** (position)* Genotype (n = 133) (n =233) (n = 164) HPC Sporadic All cases PROd AA 0.79 0.75 0.85 (−14,742bp) AG 0.18 0.22 0.14 GG 0.03 0.03 0.01 0.15 0.013 0.011 INDEL1 11 0.780.75 0.85 (−14,456 bp) 12 0.20 0.23 0.13 22 0.02 0.02 0.02 0.16 0.040.04 15b CC 0.94 0.85 0.91 (22,788 bp) CA 0.06 0.13 0.09 AA 0 0.02 00.46 0.04 0.19 P275A CC 0.88 0.92 0.81 (22,850 bp) CG 0.12 0.08 0.18 GG0 0 0.01 0.06 0.007 0.004 16a CC 0.95 0.92 0.96 (27,565 bp) CT 0.05 0.080.04 TT 0 0 0 0.48 0.2 0.24 INDEL7 11 0.87 0.89 0.78 (34,540 bp) 12 0.130.11 0.21 22 0 0 0.01 0.05 0.01 0.007 *Genomic DNA is based on NT_015280and the position is relative to the ATG site of MSR1 **Based on χ² ofAmitage trend tests, adjusted for age

The SNP P275A and 5 other frequently observed variants were furthergenotyped in 277 sporadic cases and 271 unaffected controls to assessassociation between the sequence variants and prostate cancer. Becausethe majority of case subjects in our study were Caucasians, thestatistical test was limited to Caucasians to decrease the impact ofpotential population stratification. All of the sequence variants werein Hardy-Weinberg Equilibrium among both cases and controls. Four ofthese sequence variants had significantly different allele frequenciesbetween cases and controls (Table 3). The frequency of the variantallele ‘G’ of P275A was significantly lower in HPC probands (6%), and insporadic cases (4%), compared with unaffected controls (10%), suggestingthis sequence variant affects prostate cancer susceptibility. However,because the exact function of MSR1, and the impact of the P275A on theMSR1 function are both unknown without further data, it is difficult toclassify whether the variant allele protects against or the wild typeallele increases risk for prostate cancer. Similar results were observedfor INDEL7, 15b, and INDEL1. Because these sequence variants are instrong linkage disequilibrium (P<0.0001 for all pair-wise LD tests), itis difficult to determine whether these sequence variants affect therisk of prostate cancer independently, or through strong linkagedisequilibrium with known or unknown variants in the gene. Collectively,the significant association results between these frequently observedSNPs, and the lack of linkage between the SNP P275A and prostate cancer,suggest that these SNPs are prevalent, yet low penetrance sequencevariants.

FIG. 3 shows total serum IgE in individuals homozygous for the wild-typeMSR1, or heterzygous for the R293X mutation. FIG. 4 shows the frequencyof the MSR1 nonsense mutation R293X in asthma patients as compared tocontrols. There is an increased frequency of the mutation in asthmapatients, particularly within the Hispanic population.

Caution should be taken when interpreting and generalizing the findingsof the case-control study. As a case-control study, the results aresubject to potential population stratification: that is, the differentgenotype frequencies observed may partially reflect different geneticbackgrounds in cases and controls. However, population stratification isunlikely to be substantial in this population because: 1) thestatistical tests were limited to Caucasian subjects only, and 2) noevidence was observed for a significant difference in the geneticbackground between cases and controls, based on a sample of 24consecutive SNPs recently genotyped on chromosomes 1, 8, 11, 12, and X(data not shown).

The spectrum of coding sequence changes observed in the prostate cancerpatients deserves comment. The MSR1 protein has six predicted proteindomains, including an amino terminal cytoplasmic domain, transmembrane,spacer, alpha helical coiled coil, collagen-like, and a cysteine richC-terminal domain (FIG. 1). These protein domains include 1) amino acids344-453 are the scavenger receptor cysteine-rich (SRCR) domain; 2) aminoacids 275-343 are the collagen-like domain; 3) amino acids 111-274 arethe α-helical coiled-coil domain; 4) amino acids 78-110 are the spacer;5) amino acids 51-77 are the transmembrane domain; and 6) amino acids1-50 are the cytoplasmic domain. The core of the ligand-binding regionis located in the lysine rich C-terminal end of the collagen-likedomain. The truncating mutation, R293X, results in deletion of most ofthe collagen-like domain, including the ligand-binding region and theentire cysteine rich domain. Interestingly, (Acton et al. (1993) J.Biol. Chem. 268, 3530-3537) demonstrated that the experimentally createdMSR1 mutant V296X, is synthesized and processed normally, appearing onthe surface of transfected COS cells; however, the protein does not bindtypical MSR1 ligands, and most importantly, has the ability to blockligand binding when expressed in the presence of wild type protein,suggesting a dominant negative phenotype. In the helical coiled coildomain of MSR1, mutagenesis studies have defined a critical heptapeptidesequence, ¹⁷³ IDEISKS, as comprising the functional “trigger”, requiredfor proper polymerization of the three MSR1 polypeptide chains (Frank etal. (2000) J. Biol. Chem. 275, 11672-11677). Within this sequence, asalt bridge formed between the ¹⁷⁴D and ¹⁷⁸K is thought to be crucial tothe activity of the ¹⁷³IDEISKS motif. The mutation observed in fourAfrican American families, replacing this ¹⁷⁴D with a ¹⁷⁴Y, mightinterfere with this activity, leading to less efficient trimerization ofMSR polypeptides. Substitutions of amino acid residues in the N-terminalcytoplasmic domain of MSR1 have been associated with impaired receptorinternalization and decreased ligand processing (Heider et al. (2001)FEBS Lett. 505, 185-190, Fong & Le. (1999) J. Biol. Chem. 274,36808-36816). More recently, this domain has been shown to specificallybind multiple cytoplasmic proteins including HSP70, HSP90 and GAPDH(Nakamura et al. (2002) Biochem. Biophys. Res. Commun. 290, 858-864),although the critical residues in these interactions have not beenmapped. Two missense mutations were found in prostate cancer families inthe highly conserved C-terminal cysteine rich domain. Scavenger receptorcysteine-rich (SRCR) domains are the eponymous subunit of the SRCRsuperfamily, which includes Type A and B domains. Despite being widelyfound in cell surface molecules and in secreted proteins, theirbiological functions are not well understood, and ligand-bindingproperties remain mostly speculative. The loss of SRCR domain in MSR1isoform II is not detrimental to ligand binding efficiency, althoughpartial loss of the domain in the type III isoform leads to ER membraneentrapment and loss of cell surface activity (Platt & Gordon. (2001) J.Clin. Invest 108, 649-654.).

The recurring P275A variant, seen in 30 HPC families, is located in thefirst G-X-Y repeat of the collagen-like domain (GP²⁷⁵P), and thisposition is conserved in all four mammalian species sequenced to date(man, mouse, cow, and rabbit). Since this sequence variant is common inthe general population, and appears to be less common in prostate cancercases, it will be of considerable interest to see if this mutation hasany effect on MSR1 function, and whether it may in fact have somestabilizing influence.

At present it is unknown how a lack of fully functional copies of MSR1might contribute to prostate carcinogenesis. Expression of MSR1 islargely restricted to macrophages, although expression has also beenreported in specialized endothelium, smooth muscle cells, and microglialcells, particularly in response to injury (Geng & Hansson. (1995) Scand.J. Immunol. 42, 289-296, Bell et al. (1994) J. Neurocytol. 23, 605-613).Several recent studies have examined the correlation between the extentof macrophage infiltration into tumor tissue and prostate cancer outcome(Shimura et al. (2000) Cancer Res. 60, 5857-5861, Lissbrant et al.(2000) Int. J. Oncol. 17, 445-451). Using immunochemical techniques ithas been demonstrated that macrophages present in both benign andcancerous prostate tissues routinely express MSR1 (CME and WBIunpublished observations). DeMarzo et al. and Nelson et al. haveobtained data that indicate that inflammation and features associatedwith this process (proliferative regeneration of prostate epithelium inthe presence of increased oxidative stress) most likely play key rolesin prostate cancer formation (De Marzo et al. (1999) Am. J. Pathol. 155,1985-1992, Nelson et al. (2001) Urology 57, 39-45). MSR1, by virtue ofits induction by oxidative stress (Mietus-Snyder et al. (1998)Arterioscler. Thromb. Vase. Biol. 18, 1440-1449), and its ability tobind oxidized LDL, may modify levels of reactive oxygen in this context.The finding that MSR1 knockout mice have a reduced capacity toeffectively eradicate certain pathogens may also be relevant (Suzuki etal. (1997) Nature 386, 292-296, Thomas et al. (2000) J. Exp. Med. 191,147-156.), as an infectious etiology of prostate cancer has long beenproposed (Strickler & Goedert. (2001) Epidemiol. Rev. 23, 144-151).Finally, the finding that most men over age 70 have lesionshistologically identifiable as prostate cancer indicates that theinitiation of the disease is quite ubiquitous (Dhom. (1983) J. CancerRes. Clin. Oncol. 106, 210-218), and suggests that inherited influenceswhich increase the rate of progression of these initiated lesions toclinically detectable disease may result in familial clustering ofprostate cancer—whether MSR1 mutations play a role in this progressivephase of the disease may be worth investigating.

Thus, the identification of multiple mutations and sequence variants inthe MSR1 gene, the co-segregation of multiple mutations with prostatecancer in HPC families, the rarity of these mutations in the generalpopulation, and the significant difference in the frequencies ofmultiple sequence variants between prostate cancer cases and controls,have provided novel genetic evidence that MSR1 may play an importantrole in both sporadic and hereditary prostate cancer susceptibility.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1. A method of screening a subject for increased risk of asthma,comprising: detecting the presence or absence of an MSR1 mutation insaid subject; and then determining that said subject is at increasedrisk of asthma due to the presence of said MSR1 mutation; said MSR1mutation selected from the group consisting of the R293X mutation, andthe P275A mutation.
 2. The method of claim 1, wherein said detectingstep is carried out by collecting a biological sample from said subject,and detecting the presence or absence of said mutation in saidbiological sample.
 3. The method of claim 1, wherein said detecting stepincludes a nucleic acid amplification step.
 4. The method of claim 1,wherein said detecting step includes a probe hybridization step.
 5. Themethod of claim 1, wherein said detecting step further comprisesdetecting whether said subject is homozygous for said MSR1 mutation. 6.A method of screening a subject for increased risk of asthma,comprising: detecting the presence or absence of an R293X MSR1 mutationin said subject; and then determining that said subject is at increasedrisk of asthma due to the presence of said R293X MSR1 mutation.
 7. Themethod of claim 6, wherein said detecting step is carried out bycollecting a biological sample from said subject, and detecting thepresence or absence of said R293X MSR1 mutation in said biologicalsample.
 8. The method of claim 6, wherein said detecting step includes anucleic acid amplification step.
 9. The method of claim 6, wherein saiddetecting step includes a probe hybridization step.
 10. The method ofclaim 6, wherein said detecting step further comprises detecting whethersaid subject is homozygous for said R293X MSR1 mutation.
 11. A method ofscreening a subject for increased risk of asthma, comprising: detectingthe presence or absence of a P275A MSR1 mutation in said subject; andthen determining that said subject is at increased risk of asthma due tothe presence of said P275A MSR1 mutation.
 12. The method of claim 11,wherein said detecting step is carried out by collecting a biologicalsample from said subject, and detecting the presence or absence of saidP275A MSR1 mutation in said biological sample.
 13. The method of claim11, wherein said detecting step includes a nucleic acid amplificationstep.
 14. The method of claim 11, wherein said detecting step includes aprobe hybridization step.
 15. The method of claim 11, wherein saiddetecting step further comprises detecting whether said subject ishomozygous for said P275A MSR1 mutation.